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Dec 1, 2015 - Andrey Belyaev , Thuy Minh Dau , Janne Jänis , Elena V. Grachova ... Laura A. Crandall , Alexander J. King , Richard S. Herrick , Victo...
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Supramolecular AuI−CuI Complexes as New Luminescent Labels for Covalent Bioconjugation Andrei A. Belyaev,† Dmitrii V. Krupenya,† Elena V. Grachova,† Vladislav V. Gurzhiy,‡ Alexei S. Melnikov,§,∥ Pavel Yu. Serdobintsev,§,∥ Ekaterina S. Sinitsyna,† Evgenia G. Vlakh,† Tatiana B. Tennikova,*,† and Sergey P. Tunik† †

St. Petersburg State University, Institute of Chemistry, Universitesky pr. 26, 198504 St. Petersburg, Russia St. Petersburg State University, Institute of Earth Sciences, 199034 St. Petersburg, Russia § St. Petersburg State University, Department of Physics, 198504 St. Petersburg, Russia ∥ Institute of Nanobiotechnologies, Peter the Great St. Petersburg Polytechnic University, 195251 St. Petersburg, Russia ‡

S Supporting Information *

ABSTRACT: Two new supramolecular organometallic complexes, namely, [Au6Cu2(C2C6H4CHO)6(PPh2C6H4PPh2)3](PF6)2 and [Au6Cu2(C2C6H4NCS)6(PPh2C6H4PPh2)3](PF6)2, with highly reactive aldehyde and isothiocyanate groups have been synthesized and characterized using X-ray crystallography, ESI mass spectrometry, and NMR spectroscopy. The compounds obtained demonstrated bright emission in solution with the excited-state lifetime in microsecond domain both under single- and two-photon excitation. The luminescent complexes were found to be suitable for bioconjugation in aqueous media. In particular, they are able to form the covalent conjugates with proteins of different molecular size (soybean trypsin inhibitor, human serum albumin, rabbit anti-HSA antibodies). The conjugates demonstrated a high level of the phosphorescent emission from the covalently bound label, excellent solubility, and high stability in physiological media. The highest quantum yield, storage stability, and luminance were detected for bioconjugates formed by covalent attachment of the aldehyde-bearing supramolecular AuI−CuI complex. The measured biological activity of one of the labeled model proteins clearly showed that introduced label did not prevent the biorecognition and specific protein−protein complex formation that was extremely important for the application of the conjugates in biomolecular detection and imaging.

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INTRODUCTION Currently, luminescent methods have become the most useful and powerful techniques in life sciences and related areas.1 The application of luminescent markers allows for sensitive detection with extraordinarily high efficiency both in vitro and in vivo. Proteins, nucleic acids, and other biomolecules can be conjugated with luminescent probes to give highly receptive reagents for numerous in vitro assays. Moreover, luminescent labels demonstrate enormous advances for visualization of, for example, living cells and tissue samples. Among the existing luminescent compounds, organometallic complexes based on transition metals display two positive features over fluorescent organic dyes. First of all, the absorption−emission characteristics of such substances can be easily tuned via variations in ligand environment and the nature of the coordinating metal ion. Second, bioimaging with this type of luminophore allows for higher sensitivity through timegated detection of signals, which makes it possible to cut off emission of the biotissues under study.2 Furthermore, it has been found that very often transition metal complexes display © 2015 American Chemical Society

higher photostability and may be used for long-term monitoring of dynamic systems, and also for detection of low intensity signals in pulsed-acquisition mode. The candidates for application in life science have to match a number of requirements, e.g., high extinction coefficient in the visible range and high emission quantum yield, high chemical and photostability, the presence of functional groups suitable for covalent conjugation, as well as desirable solubility in water.3 Many classes of transition metal compounds meet these criteria but they usually suffer from low solubility in aqueous solutions because of the hydrophobic ligand environment and poor stability in biosystems. To increase their biocompatibility and cellular uptake, synthesis of covalent4−10 or noncovalent11−17 conjugates with peptides and proteins is now widely used. Received: October 15, 2015 Revised: November 24, 2015 Published: December 1, 2015 143

DOI: 10.1021/acs.bioconjchem.5b00563 Bioconjugate Chem. 2016, 27, 143−150

Article

Bioconjugate Chemistry Scheme 1. Synthesis of Complexes 1 and 2

conjugates were prepared and purified using protocol developed earlier but modified, and their photophysical properties were studied and compared to those obtained for the initial complex. The biological activity of conjugates and initial proteins was also compared to evaluate the influence of organometallic compound on biorecognition followed by specific biocomplementary complex formation.

The essential advantage of covalent over noncovalent attachment is related to the stability of the bioconjugates formed. To bind luminescent organometallic compound to bioactive molecules, the label used has to contain a reactive functionality allowing the conjugation under mild conditions without elimination of toxic byproducts. Formation of covalent bioconjugates with luminescent coordination complexes can be done using active functionalities of biomolecules such as lysine amino groups8−10,18 or sulfhydryl moiety of cysteine residues6 which readily react with isothiocyanate, carboxyl, aldehyde, iodoacetamide, and epoxide substituents introduced into coordinated ligands.2,19,20 The conjugation drastically changes the physicochemical properties of the starting luminescent compounds to enhance their solubility and stability in biological systems without substantial changes of their emission characteristics. It has to be mentioned that, in many cases, the conjugates obtained display highly selective binding to certain bioobjects4 due to the specific nature of the biological unit of the aggregate. This allows for application of these types of conjugates as selective luminescent labels and extremely specific dyes for staining certain tissues and cellular organelles in bioimaging. One of the critically important drawbacks of the phosphorescent labels is strong (typically order of magnitude) quenching of their luminescence due to interaction with the triplet ground state of molecular oxygen. To increase emission of the phosphorescent dyes in in vitro experiments it is necessary to remove oxygen from the system under study, that makes these measurements much more complicated and often not possible in vivo. Earlier we reported on the synthesis of a series of heterometallic Au−Cu and Au−Ag complexes, which are extremely highly phosphorescent compounds under both single and double quantum excitation and also show negligible oxygen quenching.21−24 The application of this class of emitters for cell staining has also been demonstrated.25,26 High yield synthesis and exceptional stability of reaction products allows for easy functionalization of alkynyl ligand to vary their chemical and spectroscopic properties.24,27,28 Recently, we reported on synthesis and characterization of AuI−CuI supramolecular complex containing succinimide activated ester groups as potential label for preparation of covalent bioconjugates.29 To extend our knowledge about the chemistry and properties of phosphorescent gold−copper supramolecular complexes, in the present work we describe the synthesis and characterization of two new compounds bearing aldehyde and isothiocyanate groups, which are known to be convenient functionalities for effective conjugation under mild conditions. A series of protein

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RESULTS AND DISCUSSION Synthesis and Structural Characterization of AuI−CuI Supramolecular Complexes. The luminescent complexes 1 and 2 were prepared using the standard procedure described earlier for these type of complexes (Scheme 1).21,22,24 The reaction of alkynyl-gold polymer with stoichiometric amounts of the diphosphine ligand and [Cu(NCMe)4]PF6 affords the target [Au6Cu2(C2C6H4R)6(PPh2C6H4PPh2)3](PF6)2; R = CHO (1), NCS (2) complexes in acceptable yield. Compounds 1 and 2 were completely characterized by the 1H and 31P NMR spectroscopy and ESI-MS. The crystal structure of 1 was revealed by the X-ray diffraction study and the structural plot of this molecule is given in Figure 1. Similar to the previously characterized gold−copper complexes with the same structural motif,24,27,37,38 compound 1 consists of the central [Cu2(RC2AuC2R)3]− anionic cluster wrapped by the [Au3(PPh2C6H4PPh2)3]3+ “belt”. The polymetallic core is stabilized by electrostatic and metallophilic interactions. The Au−Au and Au−Cu contacts in 1 fall in the ranges typical for the congeners containing the same central cluster skeleton. The ESI mass spectra of 1 and 2 display signals of doubly charged [Au6Cu2(C2C6H4R)6(PPh2C6H4PPh2)3]2+ cations at m/z 1711.15 and 1798.52, respectively; isotopic patterns of these signals completely fit the stoichiometry of these species. The 31P and 1H NMR spectra of 1 and 2 feature spectroscopic patterns, where the number of signals, their multiplicity, coupling constants, and relative intensity are completely compatible with the structure found in the solid state. It is also worth noting that major characteristics of these spectra are nearly identical to those found earlier for the congeners containing different alkynyl substituents.24,27,29,38 These observations indicate that the compounds obtained are sufficiently stable in solution and retain the general structural motif of this type of complexes that in turn results in generation of effective emission (vide infra), which is essentially determined by the presence of the polynuclear heterometallic cluster core. Photophysical Properties of Complexes 1 and 2. The absorption spectra of compounds 1 and 2 (Figure 2) are substantially similar due to the similarity of the chromophoric 144

DOI: 10.1021/acs.bioconjchem.5b00563 Bioconjugate Chem. 2016, 27, 143−150

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Bioconjugate Chemistry

Table 1. Photophysical Properties of 1 and 2 in CH2Cl2 Solution (Room Temperature, λexit = 351 nm)

1 2 a

λabs, nm (ε, ×103 M−1 cm−1) [Ba, ×103 M−1 cm−1]

λem, nm

τ, μs

Q.Y., %

266 (85) [45], 323 (48) [25], 355sh (37) [20], 411 (22) [12], 460sh (9) [4.9] 266 (78) [18], 317 (65) [15], 355sh (42) [9.7], 410 (26) [6.0], 460sh (12) [2.8]

587

2.5 ± 0.1

54 ± 6

596

2.4 ± 0.1

23 ± 3

Brightness of complexes 1 and 2.

the microsecond time domain together with large Stokes shift indicate a phosphorescence origin of the emission. The position of the emission bands falls in the range typical for the complexes of this sort; 1 also displays a blue shift of emission maximum compared to 2 that is in line with the previously found trend related to the effect of electron donor properties of the akynyl ligand substituents.24 Quantum yield and emission lifetimes were measured in air-saturated solutions to mimic the conditions of imaging experiments. It is also worth noting that previous photophysical studies of this type of complex demonstrated negligible oxygen quenching of emission due to isolation of chromophoric centers by the ligand environment that determines the prospective application in imaging. Two Photon Absorption (TPA) and Two Photon Emission (TPE) Properties of 1 and 2. It has been found that similar to the other heterometallic supramolular cluster complexes of this type23,26 compounds 1 and 2 display two photon absorption under excitation in the NIR region. To track changes in the photophysical properties of these complex objects and dependence of TPA and TPE properties of the substituent in the alkynyl ligands, we also studied the previously synthesized AuI−CuI supramolecular complex containing succinimide activated ester groups29 (compound 3). Quantitative measurements of δTPE and δTPA were performed as described previously.26 Here we give just a brief description of the protocol. TPA cross sections were measured by the two photon induced fluorescence method.40 Coumarin 307 and Fluorescein were used as reference dyes.41,42 We estimate the relative measurement uncertainty of 10%, and the absolute value of the cross sections uncertainty of 30%. It is worth noting that emission maxima in TPE spectra of 1−3 are exactly the same as those found under single-photon excitation (see Figure S2). Emission intensity displays quadratic dependence on the incident laser power (Figure S3) for all substances that is indicative of the expected second-order nonlinear process and allows measurements of TPE and calculation of TPA cross sections, which are given in Table 2. The analysis of the TPA cross-section dependence on the excitation wavelengths (see Figure 3) showed that both complexes may generate TPE under excitation in the 740− 850 nm range. The value of δTPA reaches the magnitude ca. 220 GM for 1 and 308 GM and 2 at 730 nm, which is comparable

Figure 1. Molecular image of the dication 1. Hydrogen atoms of phosphine phenyl rings are omitted for clarity. Selected interatomic distances (Å): Au1−Cu1 2.834(2), Au3−Cu1 2.891(2), Au5−Cu1 2.750(2), Au1−Cu2 2.917(2), Au3−Cu2 2.754(2), Au5−Cu2 2.792(2), Au1−Au3 3.284(1), Au3−Au5 3.3817(9), Au5−Au1 3.3391(7),, Au1−Au2 2.8634(9), Au3−Au4 2.864(1), Au5−Au6 2.8955(7), Au2−P2 2.3181(36), Au2−P1 2.3300(35), Au4−P4 2.3132(35), Au4−P3 2.3190(37), Au6−P5 2.3149(43), Au6−P6 2.3170(43).

Figure 2. Absorption and emission spectra of 1 and 2 in dichloromethane solution (room temperature, λexit = 351 nm).

centers localized in the heterometallic cluster core and only slightly perturbed by peripheral ligand substituents. As previously ascribed,24,39 the high-energy absorption bands at 260−350 nm correspond to intraligand π → π* transitions of the alkynyl moieties and transitions from the σ(Au−P) orbitals to the π*(CC or phosphine) orbitals. The low-energy band at 410 nm with the shoulder stretched up to 500 nm is assigned to d(AuCu) → π*(alkynyl) charge transfer transitions. Like most previously synthesized compounds of this family,24,27,29,37,38 1 and 2 display strong emission in a dichloromethane solution. Table 1 summarizes the spectroscopic characteristics of the complexes 1 and 2. The lifetimes in

Table 2. TPA and TPE Properties of 1−3 in Acetone Solution (λexit = 790 nm) complex 1 2 3 145

δTPA, GM 71 104 55

δTPE, GM

Q.Y., %

14.5 14.3 15.0

20 ± 2 14 ± 2 27 ± 3

DOI: 10.1021/acs.bioconjchem.5b00563 Bioconjugate Chem. 2016, 27, 143−150

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Bioconjugate Chemistry

Table 3. Dependence of Amount of Conjugated Luminescent Label on Protein-to-Metal Organic Complex Ratioa complex 1

protein SBTI SBTI HSA HSA AntiHSA AntiHSA AntiHSA

Figure 3. TPA cross-section dependence on the excitation wavelengths for 1−3 in acetone solution.

to commercially available organic fluorophores, i.e., δTPA is ∼250 GM for Rhodamine B.42 Synthesis of Bioconjugates. The both types of alkynyl ligand substituents in 1 and 2 can react directly with aminobearing biomolecules to form covalent conjugates. These reactions are widely used in bioconjugate preparation.43 In particular, aldehyde groups can react with amines through a formation of Schiff’s base as intermediate. The interaction is pH dependent, being more efficient at low pH and especially efficient under high pH conditions. Since Schiff’s base is rather labile bond, it can be chemically stabilized by reduction to give a secondary amine linkage between the conjugated molecules. In our work the reaction of conjugation was carried out at pH 8.0 for 60 min followed by reduction of imine bonds (Schiff’s base) and unreacted aldehyde groups of 1 with sodium borohydride. In the case of isothiocyanate function, the conjugation reaction with the substrates containing primary amino groups, a stable reaction product was formed. Isothiocyanate containing compounds are almost entirely selective for binding with εamino groups in lysine side chains and N-terminal α-amines in proteins.44 It is well-known that isothiocyanate groups react most efficiently at alkaline pH values (pH 9.0),43 where the target amine groups are unprotonated. According to the published data, the conjugation of isothiocyanate-bearing complex 2 was carried out at pH 9.0 for 60 min. Several proteins of different molecular mass: trypsin inhibitor (SBTI, FW 20 100), human serum albumin (HSA, FW 66 400), and anti-HSA antibody (anti-HSA, FW 150 000) were used as model biomolecules for conjugation with 1 and 2. To study the effect of protein-to-label ratio on the bioconjugation process, various excess of organometallic complexes was used in the reaction. It is known that the amount of bound label is directly dependent on protein size. In general, the bigger the protein, the greater the amount of labels that can be conjugated with biomolecule avoiding its inactivation. Thus, for the conjugation of 1 and 2 with small enough proteins such as SBTI and HSA the protein/label ratio was not higher than 1/5, while for such large protein as anti-HSA antibody this ratio was increased up to 1/10. Table 3 illustrates the initial protein/complex ratio and the amount of organometallic complexes 1 and 2 bound to the protein molecules. The quota of the conjugated organometallic complex 1 was in the range of 64−70%, whereas for complex 2 this ratio was slightly less (57−66%).

complex 2

quota of conjugated complex, %

calculated protein/ complex ratio, mol/mol

quota of conjugated complex, (%)

1.0/2.1 1.0/3.2 1.0/1.8 1.0/3.2 1.0/3.3

70 64 60 64 66

1.0/1.7 1.0/2.9 1.0/1.8 1.0/3.3 1.0/3.0

57 58 60 66 60

1/7

1.0/4.5

64

1.0/4.0

57

1/10

1.0/6.7

67

1.0/6.0

60

initial protein/ complex ratio, mol

calculated protein/ complex ratio, mol/mol

1/3 1/5 1/3 1/5 1/5

a Conditions: reaction media: complex 1, 0.01 M sodium borate buffer, pH 8.0; complex 2, 0.1 M sodium borate buffer, pH 9.0; reaction time, 60 min; separation of the bioconjugates obtained from unbound luminescent label was carried out by gel-filtration using Sephadex G100 column; calculation was performed on UV analysis data at 265 nm.

Photophysical Properties of Conjugates. The conjugates of 1 and 2 with SBTI, HSA, and anti-HSA antibody display bright luminescence and their photophysical properties under one photon excitation are summarized in Table 4. Table 4. Photophysical Properties of the Conjugates with SBTI, HSA, and anti-HSA Antibody in Borate Buffer Solution (Room Temperature, λexit = 351 nm) λabs, nm (ε, ×103 M−1 cm−1)

λem, nm

1

267 (38), 342 (16), 411 (11)

593

1-HSA

267 (60), 339 (26), 412 (14)

593

1-SBTI

268 (43), 340 (20), 415 (11)

593

1-antiHSA 2

268 (89), 338 (39), 414 (27)

593

265 (19), 364 (5.8), 411 (3.9)

610

2-HSA

264 (38), 329 (14), 362 (11), 409 (7.4) 266 (32), 329 (12),364 (9.6), 407 (6.3) 267 (79), 329 (26), 410 (14)

620

2-SBTI 2-antiHSA

618 624

τ, μs (Pa) 0.9 (0.51), 2.6 (0.49) 0.8 (0.59), 2.2 (0.41) 0.7 (0.59), 2.0 (0.41) 0.6 (0.59), 2.0 (0.41) 0.039(0.6), 0.20(0.4) 0.041(0.67), 0.20(0.23) 0.035 (0.63), 0.17 (0.27) 0.045 (0.4), 0.16 (0.6)

a

The relative contribution of the components in the afterglow; for details see SI.

Absorption and emission spectra of the conjugates of 1 are shown in Figure 4; the corresponding spectra for the conjugates of 2 and all excitation spectra of the conjugates are presented in Figures S4 and S5. Table 4 and these figures also contain the data for aqueous buffer solutions of 1 and 2 to elucidate the solvent influence on their photophysical properties. Absorption and excitation spectra of initial complexes and their conjugates look essentially similar which is indicative of the preservation of the chromophoric centers in the course conjugation. The emission of 1 in the borate buffer solution is slightly red-shifted (∼5 nm) compared to emission maximum 146

DOI: 10.1021/acs.bioconjchem.5b00563 Bioconjugate Chem. 2016, 27, 143−150

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Bioconjugate Chemistry

To characterize quantitatively the affinity pair formation, the inhibition constants (Ki) were calculated using Dixon’s plots. As seen from Figure 5, Ki values for 1-SBTI and 2-SBTI were found to be 160 and 145 nM, respectively. These values were

Figure 4. Absorption and emission spectra of 1 and its conjugates with proteins in borate buffer solution (room temperature, λexit = 351 nm).

in dichloromethane solution that may be explained by increasing polarity of the media. The conjugation of 1 with proteins does not give any changes in the position of emission maximum. The emission of 2 in the buffer is also red-shifted for ∼15 nm compared to the dichloromethane solution, and depending on the nature of protein in the conjugates, one can observe the further red shift of the luminescence band. The emission of 1 in the borate buffer shows double exponential decay with 0.9 and 2.6 μs components; the long one is very close to that observed in dichloromethane and the other is very probably caused by emission quenching with water molecules. The emission lifetimes in the conjugates of 1 also show double exponential decay with slightly different lifetimes of both components. Complex 2 demonstrates much higher sensitivity with respect to emission quenching in buffer solution to give substantially shorter lifetimes and evidently lower emission intensity. We have found that the conjugates of biomolecules with 1 are surprisingly high stable. The emission spectrum and intensity of luminescence (with an accuracy of about 10%) did not change for 45 days (see Figures S6, S7). Unfortunately, we failed to measure correctly the luminescence quantum yield of the bioconjugates with 1−3 due to high scattering in the solutions studied. This also prevents TPA measurements for the conjugates obtained. Therefore, their absolute brightness was measured by direct comparison of conjugates photophysical properties (Figure S8). Biological Activity of Luminescent Label−Protein Conjugates. Since the studied compounds 1 and 2 are large with molecular weight about 4000, it was important not only to conjugate them with biomolecules, but also to save their photophysical properties, as well as the biological activity of the conjugates formed. It is reasonably to presume that small proteins are more sensitive to the intervention into their tertiary structure in comparison to large biomolecules. Moreover, besides the simple inactivation because of distortion of the protein active center, some steric hindrance may also take place at the interaction of labeled protein with its natural counterpart. To evaluate the influence of big organometallic compounds covalently bound to the protein on the specific protein−protein interactions, the interactions between the conjugates of small protein SBTI (1-SBTI and 2-SBTI) and its affinity partner trypsin were investigated. The formation of specific complex SBTI-trypsin followed by reduction of trypsin activity45 could be monitored as decreasing of the rate of enzymatically catalyzed digestion of specific substrate, BAPNA.

Figure 5. Dixon’s plots of trypsin inhibition by conjugate of SBTI with organometallic complex 1 (A) and 2 (B).

about two times higher compared to the Ki of native inhibitor (88 nM). The calculated values of Ki for conjugated SBTI allow the conclusion that the discussed luminescent complexes changed the protein activity insignificantly. According to Table 5, Michaelis−Menten’s constant (KM) and specific activity (Asp) for BAPNA hydrolysis without Table 5. Effect of Inhibition on Trypsin Activity for BAPNA Hydrolysisa BAPNA trypsinolysis Without inhibition In presence of native SBTI In presence of 1-SBTI In presence of 2-SBTI

KM, mM

specific activity, μmol·min−1·mg−1

0.37 ± 0.02 0.34 ± 0.01

1.01 ± 0.04 0.54 ± 0.02

0.35 ± 0.02 0.34 ± 0.03

0.58 ± 0.03 0.56 ± 0.03

a

Conditions: the reaction (molar) ratio SBTI/metal organic complex was equal to 1/3; the ratio trypsin to SBTI was 1.00/0.45.

inhibitor, in the presence of native and conjugated SBTI with 1 and 2 were strictly coincided that confirmed the similar behavior of both native and labeled forms of SBTI.

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CONCLUSIONS Two new supramolecular AuI−CuI complexes bearing the reactive aldehyde or isothiocyante functionalities with intensive phosphorescence in the solution both under single- and twophoton excitation were synthesized and characterized. A possibility of their application for bioconjugation was also 147

DOI: 10.1021/acs.bioconjchem.5b00563 Bioconjugate Chem. 2016, 27, 143−150

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Bioconjugate Chemistry

amorphous brown solid and used without further purification. Yield: 81% (276 mg). [Au6Cu2(AuCCC6H4-4-CHO)6(dppb)3](PF6)2 (1). [AuC CC6H4-4-CHO]n (82 mg, 0.25 mmol) was suspended in 5 mL of dichloromethane and dppb (56 mg, 0.125 mmol) was added. The reaction mixture was stirred for 30 min to become transparent and [Cu(NCMe)4]PF6 (31 mg, 0.08 mmol) was added. After stirring for an additional 30 min the reaction mixture turned orange-red. The solution obtained was filtered through Celite and dried under vacuum. The product was obtained as orange crystals by gas phase diffusion of pentane into dichloromethane solution. Yield: 91% (141 mg). ESI-MS: m/z = 1711.15 [M]2+ (calc.1711.14). 31P{1H} NMR (CDCl3, RT) δ: 44.6 (s, 6P), −144.8 (sept, 1JPF = 705 Hz, 2P, PF6). 1H NMR (CDCl3, RT) δ: 9.83 (s, 6H, CHO), 8.02 (dm, 3JHH = 7 Hz, 3JHH = 14 Hz, 24H, ortho-H), 7.88 (m, 12H, P−C6H4−P), 7.67 (t, 3JHH = 7.4 Hz, 12H, para-H), 7.49 (dd, 3JHH = 7.4 Hz, 3 JHH = 8.4 Hz, 24H, meta-H), 7.35 (d, 3JHH = 8.4 Hz, 12H, CC−C6H4), 7.02 (d, 3JHH = 8.4 Hz, 12H, CC−C6H4). [Au 6Cu2(CCC6H4-4-NCS)6(dppb)3](PF6)2 (2). [AuC CC6H4-4-NCS]n (70 mg, 0.20 mmol) and dppb (43 mg, 0.10 mmol) were dissolved in dichloromethane (20 mL) and stirred for 24 h. The reaction mixture was filtered and a solution of [Cu(NCMe)4]PF6 (20 mg, 0.056 mmol) in 2 mL of dichloromethane was added. The reaction mixture was stirred for 2 h while it turned orange. The product was obtained as orange crystals by gas phase diffusion of ether into dichloromethane solution. Yield: 50 mg (43%). ESI-MS: m/z = 1798.52 [M]2+ (calc.1798.55). 31P{1H} NMR (acetone-d6, RT) δ: 44.6 (s, 6P), −144.8 (sept, 1JPF = 705 Hz, 2P, PF6). 1H NMR (acetone-d6, RT) δ: 7.87 (dm, 3JHH = 7 Hz, 3JHH = 13 Hz, 24H, ortho-H), 7.58 (m, 24H, P−C6H4−P, para-H), 7.41 (m, 24H, meta-H), 6.67 (d, 3JHH = 7.4 Hz, 12H, CC−C6H4), 6.56 (d, 3 JHH = 7.4 Hz, 12H, CC−C6H4). X-ray Crystal Structure Analysis. The crystal structure of 1 was determined by the means of single crystal X-ray diffraction analysis. A crystal of 1 was fixed onto a micromount, placed on an Agilent Technologies Supernova Atlas diffractometer, and measured at a temperature of 100 K using microfocused monochromated Cu Kα radiation. The unit cell parameters and refinement characteristics for the crystal structure of 1 are given in Table S1. The unit cell parameters were determined and refined by the least-squares techniques on the basis of 52 801 reflections with 2θ in the range of 5.85− 135.00°. From the systematic absences and statistics of reflection distribution, the space group P1̅ was determined. The structure was solved by direct methods and refined to R1 = 0.085 (wR2 = 0.167) for 17152 reflections with |Fo| ≥ 4σ F using the SHELXL-97 program34 incorporated in the OLEX2 program package.35 Empirical absorption correction for 1 was applied in CrysAlisPro36 program complex using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm. The carbon-bound H atoms were placed in calculated positions and were included in the refinement in the “riding” model approximation, with Uiso(H) set to 1.5Ueq(C) and C−H 0.96 Å for the CH3 groups, Uiso(H) set to 1.2Ueq(C) and C−H 0.97 Å for the CH2 groups and Uiso(H) set to 1.2Ueq(C) and C−H 0.93 Å for the CH groups. It should be noted that there are rather high peaks of the residual density near the Au atoms and some of the refinement parameters are also rather high due to the low quality of crystals and its low diffraction ability. Supplementary crystallographic data for this paper have been deposited at the Cambridge Crystallographic

demonstrated. It was found that covalent attachment of the complexes to the proteins of various molecular masses and biological nature does not substantially change the photophysical properties of the starting organometallic compounds. Moreover, despite the rather big size of these organometallic complexes, the formation of their conjugates with the proteins studied did not prevent its specific interaction with affinity partner. Thus, the organometallic compounds obtained can be used as phosphorescent labels in various diagnostics or analytical arrays based on formation of affinity complexes or other techniques proposed for detection of target biomolecule using emission signal measurement. Among the complexes studied, the aldehyde-bearing supramolecular AuI−CuI compound was found to be preferable for bioconjugation from the viewpoint of its photophysical properties, high stability, and luminance in aqueous solution.

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EXPERIMENTAL SECTION Material and Reagents. Tetrahydrothiophene (THT), 4ethynylbenzaldehyde were purchased from Sigma-Aldrich (Germany). 1-Ethynyl-4-isothiocyanatobenzene,30 1,4-bis(diphenylphosphino)benzene (dppb),31 [Au(THT)Cl],32 [Cu(NCMe)4]PF633 were synthesized according to published procedures. Soybean trypsin inhibitor (SBTI), human serum albumin (HSA), and antibodies against this protein (anti-HAS abs) were purchased from Sigma-Aldrich (Germany). All salts used for preparation of buffer solutions were produced by Fluka and Sigma-Aldrich (Germany) and had analytical-grade purity. Before use, all buffers were filtered using membranes with pore diameter of 0.45 μm (Millipore, Austria). A glass column of 280 × 5 mm i.d. packed with Sephadex G-100 (Pharmacia, Sweden) was applied to purify the bioconjugates from the excess luminescent complex (label). Instrumentation. 1H and 31P NMR spectra were recorded on Bruker DPX 300 and Bruker Avance 400 spectrometers. Mass spectra were obtained on Bruker APEX-Qe ESI FT-ICR instrument. Elemental microanalysis was carried out using EuroVector 3000. To purify the bioconjugates by gel-filtration, a low-pressure Masterflex Console Drive Easy-Load II Model 77201−60 pump (Cole-Parmer Instrument Company) chromatographic system with a UV detector 2138 Uvicord S (LKB) was used. Lyophilization of bioconjugates was performed using FreeZone1 LABCONCO. Determination of absorbance of analyzed solutions was performed using an UV−vis spectrometer UVmini-1240. Synthesis. [AuCCC6H4-4-CHO]n. 4-Ethynylbenzaldehyde (50 mg, 0.4 mmol) was dissolved in acetone (3 mL) and added to a solution of [Au(THT)Cl] (125 mg, 0.385 mmol) in acetone (5 mL). A solution of CH3COONa (0.2 g) in 5 mL of water was added to the resulting solution and the reaction mixture was stirred for 30 min in the absence of light. Greenish precipitate was collected by centrifugation, washed with water, ethanol, and diethyl ether, and dried under a vacuum. The product was obtained as amorphous brown solid and used without further purification. Yield: 72% (90 mg). [AuCCC6H4-4-NCS]n. 1-Ethynyl-4-isothiocyanatobenzene (140 mg, 0.88 mmol) was dissolved in acetone (10 mL) and [Au(THT)Cl] (300 mg, 0.95 mmol) and triethylamine (1 mL) were added. The reaction mixture was stirred for 2 h and solvents were evaporated to dryness. A brown residue was successively treated with ethanol, acetone, and pentane and dried under vacuum. The product was obtained as an 148

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Bioconjugate Chemistry Data Centre (CCDC 1051421) and can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif. Preparation of Bioconjugates. To obtain the conjugates of proteins with the synthesized organometallic complexes 1 and 2, three model proteins, namely, SBTI, HSA, and anti-HSA antibodies, were chosen. 0.01 M sodium phosphate buffers, pH 7.4 and 8.0, as well as 0.01 M sodium borate buffers, pH 8.4 and 9.0, were used as the reaction media. The protein/luminescent complex molar ratio was changed in the range from 1/3 up to 1/10 depending on protein molecular size (mass). The following concentrations of initial compounds were used for bioconjugate preparation: proteins − 1 mg/mL in chosen buffer; luminescent marker (organometallic complex) − 8 mg/ mL in acetone. The conjugates were prepared by addition of corresponding aliquot of label solution to the protein. The reaction mixture was stirred and incubated in the dark at 22 °C for time varying from 15 to 120 min. The product formation was monitored using gel-filtration on a glass column (280 × 5 mm i.d.) packed with swollen Sephadex G-100 (see Supporting Information Figure S1). The amount of loaded sample was equal to 0.1 mL; flow rate was 0.25 mL/min; the detection of all components was performed photometrically at 280 nm wavelength. After completion of conjugation, the reduction of the aldimine bonds formed, as well as unreacted aldehyde groups, was carried out with aqueous sodium borohydride (2 mg/mL) for 30 min at ambient temperature. Study of Inhibitory Ability of Labeled SBTI. The conjugates of SBTI with luminescent complexes were obtained using 1/3 molar ratio of protein to label. The reaction between SBTI and aldehyde-bearing label (complex 1) was carried out in 0.01 M Na-borate buffer, pH 8.0, stirring for 15 min. The conjugation of SBTI with isothiocyanate label (complex 2) was performed using 0.01 M Na-borate buffer, pH 8.0, stirring for 60 min. The study of biological activity of bioconjugates was performed according to the protocol published earlier.29 Photophysical Measurements. A DPPS pulse laser DTL399QT (wavelength 351 nm, pulse energy 50 μJ, repetition rate 1 kHz, pulse width 6 ns; Laser Export, Russia) was used to pump luminescence. A digital oscilloscope Tektronix TDS3014B (bandwidth 100 MHz;Tektronix, USA), a monochromator MUM (interval of wavelengths 10 nm; LOMO, Russia) and a photomultiplier tube (Hamamatsu, Japan) were used for lifetime measurements. Emission spectra were recorded using an HR2000 spectrometer (Ocean Optics Inc., USA). A halogen lamp LS-1-CAL and a DH2000 deuterium lamp (Ocean Optics Inc., USA) were used to calibrate the absolute spectral response of the spectral system in the 200− 1100 nm range. Absorption spectra were measured on a Varian Cary 50 spectrophotometer (Varian, USA). Excitation spectra were recorded on a Varian Cary Eclipse spectrofluorimeter (Varian, USA). A mode-locked Ti:sapphire laser (Avesta TiF-50F, Russia) was used for excitation two photons luminescence. Parameters of laser beam were the pulse width 50 fs, repetition rate 80 MHz, average power 450 mW, pulse energy ∼5 nJ, tuning range of wavelengths 730−870 nm.

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Crystallographic data for 1; chromatograms illustrating the separation of formed conjugates; figures illustrating the photophysical properties of the conjugates; comments to Table 4 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research related to the synthesis, characterization, and photophysical investigation of organometallic complexes was supported by the grants of St. Petersburg State University and Russian Foundation for Basic Research (## 0.37.169.2014 and 13-04-40342/13-04-40343, respectively). The part of work on bioconjugation and investigation of bioconjugate properties was granted by Russian Science Foundation (# 14-50-00069). The work was carried out using equipment of the Analytical Centre for Nano- and Biotechnologies (Peter the Great St. Petersburg Polytechnic University; with financial support from Ministry of Education and Science of Russian Federation); and Centres for Magnetic Resonance, for Optical and Laser Materials Research, and Physical methods of surface investigation (Research park of St. Petersburg State University).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00563. 149

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Bioconjugate Chemistry

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DOI: 10.1021/acs.bioconjchem.5b00563 Bioconjugate Chem. 2016, 27, 143−150